We propose to study the replication of the resistance plasmid R100 in E. coli K 12. The research is aimed at genetic and biochemical analysis of genes which are utilized in the replication of R 100. We wish to first analyze the nucleotide sequence of a region 2.5 kilobase pairs in length, which has been shown to be essential for the autonomous replication of R100, by using the Maxam and Gilbert technique. The region must contain the origin of replication, promotor(s) and structural genes which regulate the replication of the plasmid. Secondly, we plan to do extensive genetics on R100, by isolating mutants defective in replication and characterizing these mutants by complementation analysis to determine the structural and regulatory genes encoded by R100. Thirdly, we are planning a thorough biochemical analysis to identify and characterize the RNA transcripts and proteins synthesized at the essential region for the replication of R100. Analysis of the nucleotide sequences of mutants having particular cis-dominant mutations and analysis of the transcripts and proteins will clarify the arrangement of genes on the determined nucleotide sequences of the parent. Finally, we will establish an in vitro system of replication of the plasmid. The in vitro system will have great advantages to follow the specific steps of the biochemical reactions involved in replication. We wish to use this system to analyze the roles of the plasmid proteins in replication of this plasmid. Using mutant plasmids to be isolated, we expect to perform complementation analysis in vitro for plasmid DNA replication. Our proposed systematic research on the replication of R100 plasmid will elucidate many of the molecular mechanisms involved in the replication of this plasmid.